Transcription, the transfer of genetic information from DNA to RNA, is the first step of gene expression. RNA transcripts are generated by the catalytic core of the DNA dependent RNA polymerase (RNAP), which is highly conserved among all cellular organisms. In bacteria, the core RNAP interacts with the sigma factor to form the holoenzyme that specifically binds to promoter DNA sequences located upstream of transcription start sites. At most bacterial promoters, the transcription-competent RNAP holoenzyme open promoter complex (RPo) forms spontaneously from the initial closed promoter complex (RPc) through a pathway that includes transient intermediates (RPi). This pathway from RPc to RPo is the target of many transcriptional regulators that enable selective expression of certain genes in response to environmental stress. While detailed structural models of RPo reconcile the results of many biochemical and biophysical experiments, these have not addressed important questions regarding the mechanism of RPo formation nor its regulation. Recent studies identified CarD as an essential RNAP binding protein that regulates rRNA transcription in the pathogen Mycobacterium tuberculosis, but the molecular mechanism for CarD function is unknown. This proposal aims to address questions regarding the detailed mechanism of RPo formation, and the regulation of transcription initiation by CarD through X-ray crystallographic structure determination of relevant macromolecular complexes. Specifically, we propose to: 1) structurally characterize intermediates (RPi) on the pathway of RPo formation. Using novel DNA constructs, we have determined preliminary crystal structures that show expected features of intermediates. Further structural and biochemical analyses will be conducted to determine RPi model and validate its relevance. The information obtained will provide insight into this key step of transcription initiation~ and 2) Use a combination of structural and functional approaches to elucidate the molecular mechanism for the regulation of transcription initiation by CarD. Structural and detailed biochemical studies of T. thermophilus RNAP transcription initiation complexes with T. thermophilus CarD will be conducted. The results of these studies will be combined with in vivo studies of M. tuberculosis CarD (from collaborators) to provide a comprehensive picture of CarD in vitro mechanism and in vivo function.